出品公司: | Clontech |
---|---|
载体名称: | pAmCyan1-N1 |
质粒类型: | 哺乳动物细胞表达载体;荧光报告载体 |
高拷贝/低拷贝: | 高拷贝 |
克隆方法: | 限制性内切酶,多克隆位点 |
启动子: | CMV IE |
载体大小: | 4703 bp |
5' 测序引物及序列: | -- |
3' 测序引物及序列: | -- |
载体标签: | AmCyan1(C-端) |
载体抗性: | 卡那霉素 |
筛选标记: | 新霉素(Neomycin) |
克隆菌株: | DH5α, HB101 |
宿主细胞(系): | 常规细胞系,293、CV-1、CHO等 |
备注: |
pAmCyan1-N1载体是青色荧光报告载体,表达C端AmCyan1融合蛋白; AmCyan1是AmCyan的突变体,与AmCyan1相比,编码序列经过了优化,更适合在哺乳动物细胞中的表达; 同时突变了两个氨基酸位点, 改变了发光特点。 |
产品目录号: | 632442 |
稳定性: | 瞬表达 或 稳表达 |
组成型/诱导型: | 组成型 |
病毒/非病毒: | 非病毒 |
买家导航
pAmCyan1-N1载体质粒基本信息
pAmCyan1-N1质粒图谱载体图谱和pAmCyan1-N1载体序列质粒序列多克隆位点信息
pAmCyan1-N1质粒载体简介
pAmCyan1-N1载体描述
pAmCyan1-N1 encodes a human codon-optimized variant of wild-type Anemonia majano cyan fluorescent protein, AmCyan1 (1). The AmCyan1 coding sequence contains a series of silent base-pair changes, which correspond to human codon-usage preferences, for optimal expression in mammalian cells (2). Additionally, an upstream sequence—located just 5' to the AmCyan1 start codon—has been converted to a Kozak consensus translation initiation site(3) to further increase the translation efficiency in eukaryotic cells. Two amino acid substitutions (Asn-34 to Ser; Lys-68 to Met) have been made to enhance the emission characteristics of AmCyan1 (excitation maximum = 458 nm; emission maximum = 489 nm).
The multiple cloning site (MCS) in pAmCyan1-N1 is positioned between the immediate-early promoter of CMV (PCMV IE) and the AmCyan1 coding sequence. Thus, genes cloned into the MCS will be expressed as fusions to the N-terminus of AmCyan1 if they are in the same reading frame as AmCyan1 and there are no intervening stop codons. The SV40 polyadenylation signals (SV40 polyA) downstream of the AmCyan1 gene direct proper processing of the 3'end of AmCyan1 mRNA.
The vector backbone contains an SV40 origin (SV40 ori) for replication in mammalian cells that express the SV40 T antigen, a pUC origin of replication (pUC ori) for propagation in E. coli, and an f1 origin (f1 ori) for single-stranded DNA production. In addition, a neomycin-resistance cassette— consisting of the SV40 early promoter (PSV40e), the neomycin/kanamycin resistance gene of Tn5 (Neor/Kanr), and polyadenylation signals from the Herpes simplex virus thymidine kinase (HSV TK poly A) gene—allows stably transfected eukaryotic cells to be selected using G418 (4). A bacterial promoter (P) upstream of this cassette drives expression of the Neor/Kanr gene in E. coli host cells, which can be selected with kanamycin.
Fusions to the N terminus of AmCyan1 retain the fluorescent properties of the native protein allowing the localization of the fusion protein in vivo . The target gene should be cloned into pAmCyan1-N1 so that it is in frame with the AmCyan1 coding sequence, with no intervening, in-frame stop codons. The inserted gene should include the initiating ATG codon. The recombinant pAmCyan1-N1 vector can be transfected into mammalian cells using any standard transfection method. If required, stable transformants can be selected using G418 (available from Clontech; Cat. Nos. 631307 & 631308). We recommend selecting mammalian cell cultures in 500–1,300 μg/ml G418, depending on the cell line. Be sure to establish a kill curve for each cell line and each lot of G418 to determine the optimal selection concentration. Unmodified (i.e., non-recombinant) pAmCyan1-N1 can also be used simply to express AmCyan1 in a cell line of interest (e.g., as a transfection marker).
Propagation in E. coli
Suitable host strains: DH5α, HB101 and other general purpose strains. Single-stranded DNA production requires
a host containing an F plasmid such as JM101 or XL1-Blue.
Selectable marker: plasmid confers resistance to kanamycin (50 μg/ml) to E. coli hosts.
E. coli replication origin: pUC
Copy number: ~500
Plasmid incompatibility group: pMB1/Col E1
pAmCyan1-N1质粒序列载体序列
LOCUS pAmCyan1-N1 4703 bp DNA SYN DEFINITION pAmCyan1-N1 ACCESSION KEYWORDS SOURCE ORGANISM other sequences; artificial sequences; vectors. FEATURES Location/Qualifiers source 1..4703 /organism="pAmCyan1-N1" /mol_type="other DNA" promoter 10..562 /label="CMV_immearly_promoter" misc_feature 65..352 /label="CAG_enhancer" misc_feature 519..539 /label="CMV_fwd_primer" promoter 520..589 /label="CMV_promoter" CDS 679..1368 /label="ORF frame 1" /translation="MALSNKFIGDDMKMTYHMDGCVNGHYFTVKGEGSGKPYEGTQTS TFKVTMANGGPLAFSFDILSTVFMYGNRCFTAYPTSMPDYFKQAFPDGMSYERTFTYE DGGVATASWEISLKGNCFEHKSTFHGVNFPADGPVMAKKTTGWDPSFEKMTVCDGILK GDVTAFLMLQGGGNYRCQFHTSYKTKKPVTMPPNHVVEHRIARTDLDKGGNSVQLTEH AVAHITSVVPF*" misc_feature 1582..1601 /label="EBV_rev_primer" /translation="MALSNKFIGDDMKMTYHMDGCVNGHYFTVKGEGSGKPYEGTQTS TFKVTMANGGPLAFSFDILSTVFMYGNRCFTAYPTSMPDYFKQAFPDGMSYERTFTYE DGGVATASWEISLKGNCFEHKSTFHGVNFPADGPVMAKKTTGWDPSFEKMTVCDGILK GDVTAFLMLQGGGNYRCQFHTSYKTKKPVTMPPNHVVEHRIARTDLDKGGNSVQLTEH AVAHITSVVPF*" rep_origin complement(1751..2057) /label="f1_origin" /translation="MALSNKFIGDDMKMTYHMDGCVNGHYFTVKGEGSGKPYEGTQTS TFKVTMANGGPLAFSFDILSTVFMYGNRCFTAYPTSMPDYFKQAFPDGMSYERTFTYE DGGVATASWEISLKGNCFEHKSTFHGVNFPADGPVMAKKTTGWDPSFEKMTVCDGILK GDVTAFLMLQGGGNYRCQFHTSYKTKKPVTMPPNHVVEHRIARTDLDKGGNSVQLTEH AVAHITSVVPF*" promoter 2136..2164 /label="AmpR_promoter" /translation="MALSNKFIGDDMKMTYHMDGCVNGHYFTVKGEGSGKPYEGTQTS TFKVTMANGGPLAFSFDILSTVFMYGNRCFTAYPTSMPDYFKQAFPDGMSYERTFTYE DGGVATASWEISLKGNCFEHKSTFHGVNFPADGPVMAKKTTGWDPSFEKMTVCDGILK GDVTAFLMLQGGGNYRCQFHTSYKTKKPVTMPPNHVVEHRIARTDLDKGGNSVQLTEH AVAHITSVVPF*" misc_feature complement(2230..2250) /label="pBABE_3_primer" /translation="MALSNKFIGDDMKMTYHMDGCVNGHYFTVKGEGSGKPYEGTQTS TFKVTMANGGPLAFSFDILSTVFMYGNRCFTAYPTSMPDYFKQAFPDGMSYERTFTYE DGGVATASWEISLKGNCFEHKSTFHGVNFPADGPVMAKKTTGWDPSFEKMTVCDGILK GDVTAFLMLQGGGNYRCQFHTSYKTKKPVTMPPNHVVEHRIARTDLDKGGNSVQLTEH AVAHITSVVPF*" misc_feature complement(2236..2451) /label="SV40_enhancer" /translation="MALSNKFIGDDMKMTYHMDGCVNGHYFTVKGEGSGKPYEGTQTS TFKVTMANGGPLAFSFDILSTVFMYGNRCFTAYPTSMPDYFKQAFPDGMSYERTFTYE DGGVATASWEISLKGNCFEHKSTFHGVNFPADGPVMAKKTTGWDPSFEKMTVCDGILK GDVTAFLMLQGGGNYRCQFHTSYKTKKPVTMPPNHVVEHRIARTDLDKGGNSVQLTEH AVAHITSVVPF*" promoter 2248..2516 /label="SV40_promoter" /translation="MALSNKFIGDDMKMTYHMDGCVNGHYFTVKGEGSGKPYEGTQTS TFKVTMANGGPLAFSFDILSTVFMYGNRCFTAYPTSMPDYFKQAFPDGMSYERTFTYE DGGVATASWEISLKGNCFEHKSTFHGVNFPADGPVMAKKTTGWDPSFEKMTVCDGILK GDVTAFLMLQGGGNYRCQFHTSYKTKKPVTMPPNHVVEHRIARTDLDKGGNSVQLTEH AVAHITSVVPF*" rep_origin 2415..2492 /label="SV40_origin" /translation="MALSNKFIGDDMKMTYHMDGCVNGHYFTVKGEGSGKPYEGTQTS TFKVTMANGGPLAFSFDILSTVFMYGNRCFTAYPTSMPDYFKQAFPDGMSYERTFTYE DGGVATASWEISLKGNCFEHKSTFHGVNFPADGPVMAKKTTGWDPSFEKMTVCDGILK GDVTAFLMLQGGGNYRCQFHTSYKTKKPVTMPPNHVVEHRIARTDLDKGGNSVQLTEH AVAHITSVVPF*" misc_feature 2477..2496 /label="SV40pro_F_primer" /translation="MALSNKFIGDDMKMTYHMDGCVNGHYFTVKGEGSGKPYEGTQTS TFKVTMANGGPLAFSFDILSTVFMYGNRCFTAYPTSMPDYFKQAFPDGMSYERTFTYE DGGVATASWEISLKGNCFEHKSTFHGVNFPADGPVMAKKTTGWDPSFEKMTVCDGILK GDVTAFLMLQGGGNYRCQFHTSYKTKKPVTMPPNHVVEHRIARTDLDKGGNSVQLTEH AVAHITSVVPF*" CDS 2599..3393 /label="ORF frame 1" /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGR PVLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDL LSSHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDE EHQGLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRY QDIALATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF*" gene 2602..3390 /label="NeoR/KanR" /gene="NeoR/KanR" /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGR PVLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDL LSSHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDE EHQGLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRY QDIALATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF*" CDS complement(2908..3444) /label="ORF frame 3" /translation="MAGWASLGRSFRTPESRSEELVKKAIEGDALRIGSGDTVKHEEA VSPFAAKLFSNITGSQRYVLIAVRHTQPATVDESRKAAIFHHDIRQAGIAMGHDEILA VGHARLEPGEQFGWREPLMLFVQIILIDKTGFHPSTCSLDAMFRLVVEWAGSRIKRMQ PPHCISHDGYFLGRSKVR*" terminator 3568..3837 /label="TK_PA_terminator" /translation="MAGWASLGRSFRTPESRSEELVKKAIEGDALRIGSGDTVKHEEA VSPFAAKLFSNITGSQRYVLIAVRHTQPATVDESRKAAIFHHDIRQAGIAMGHDEILA VGHARLEPGEQFGWREPLMLFVQIILIDKTGFHPSTCSLDAMFRLVVEWAGSRIKRMQ PPHCISHDGYFLGRSKVR*" rep_origin 3985..4604 /label="pBR322_origin" /translation="MAGWASLGRSFRTPESRSEELVKKAIEGDALRIGSGDTVKHEEA VSPFAAKLFSNITGSQRYVLIAVRHTQPATVDESRKAAIFHHDIRQAGIAMGHDEILA VGHARLEPGEQFGWREPLMLFVQIILIDKTGFHPSTCSLDAMFRLVVEWAGSRIKRMQ PPHCISHDGYFLGRSKVR*" ORIGIN 1 TAGTTATTAA TAGTAATCAA TTACGGGGTC ATTAGTTCAT AGCCCATATA TGGAGTTCCG 61 CGTTACATAA CTTACGGTAA ATGGCCCGCC TGGCTGACCG CCCAACGACC CCCGCCCATT 121 GACGTCAATA ATGACGTATG TTCCCATAGT AACGCCAATA GGGACTTTCC ATTGACGTCA 181 ATGGGTGGAG TATTTACGGT AAACTGCCCA CTTGGCAGTA CATCAAGTGT ATCATATGCC 241 AAGTACGCCC CCTATTGACG TCAATGACGG TAAATGGCCC GCCTGGCATT ATGCCCAGTA 301 CATGACCTTA TGGGACTTTC CTACTTGGCA GTACATCTAC GTATTAGTCA TCGCTATTAC 361 CATGGTGATG CGGTTTTGGC AGTACATCAA TGGGCGTGGA TAGCGGTTTG ACTCACGGGG 421 ATTTCCAAGT CTCCACCCCA TTGACGTCAA TGGGAGTTTG TTTTGGCACC AAAATCAACG 481 GGACTTTCCA AAATGTCGTA ACAACTCCGC CCCATTGACG CAAATGGGCG GTAGGCGTGT 541 ACGGTGGGAG GTCTATATAA GCAGAGCTGG TTTAGTGAAC CGTCAGATCC GCTAGCGCTA 601 CCGGACTCAG ATCTCGAGCT CAAGCTTCGA ATTCTGCAGT CGACGGTACC GCGGGCCCGG 661 GATCCACCGG TCGCCACCAT GGCCCTGTCC AACAAGTTCA TCGGCGACGA CATGAAGATG 721 ACCTACCACA TGGACGGCTG CGTGAACGGC CACTACTTCA CCGTGAAGGG CGAGGGCAGC 781 GGCAAGCCCT ACGAGGGCAC CCAGACCTCC ACCTTCAAGG TGACCATGGC CAACGGCGGC 841 CCCCTGGCCT TCTCCTTCGA CATCCTGTCC ACCGTGTTCA TGTACGGCAA CCGCTGCTTC 901 ACCGCCTACC CCACCAGCAT GCCCGACTAC TTCAAGCAGG CCTTCCCCGA CGGCATGTCC 961 TACGAGAGAA CCTTCACCTA CGAGGACGGC GGCGTGGCCA CCGCCAGCTG GGAGATCAGC 1021 CTGAAGGGCA ACTGCTTCGA GCACAAGTCC ACCTTCCACG GCGTGAACTT CCCCGCCGAC 1081 GGCCCCGTGA TGGCCAAGAA GACCACCGGC TGGGACCCCT CCTTCGAGAA GATGACCGTG 1141 TGCGACGGCA TCTTGAAGGG CGACGTGACC GCCTTCCTGA TGCTGCAGGG CGGCGGCAAC 1201 TACAGATGCC AGTTCCACAC CTCCTACAAG ACCAAGAAGC CCGTGACCAT GCCCCCCAAC 1261 CACGTGGTGG AGCACCGCAT CGCCAGAACC GACCTGGACA AGGGCGGCAA CAGCGTGCAG 1321 CTGACCGAGC ACGCCGTGGC CCACATCACC TCCGTGGTGC CCTTCTGAAG CGGCCGCGAC 1381 TCTAGATCAT AATCAGCCAT ACCACATTTG TAGAGGTTTT ACTTGCTTTA AAAAACCTCC 1441 CACACCTCCC CCTGAACCTG AAACATAAAA TGAATGCAAT TGTTGTTGTT AACTTGTTTA 1501 TTGCAGCTTA TAATGGTTAC AAATAAAGCA ATAGCATCAC AAATTTCACA AATAAAGCAT 1561 TTTTTTCACT GCATTCTAGT TGTGGTTTGT CCAAACTCAT CAATGTATCT TAAGGCGTAA 1621 ATTGTAAGCG TTAATATTTT GTTAAAATTC GCGTTAAATT TTTGTTAAAT CAGCTCATTT 1681 TTTAACCAAT AGGCCGAAAT CGGCAAAATC CCTTATAAAT CAAAAGAATA GACCGAGATA 1741 GGGTTGAGTG TTGTTCCAGT TTGGAACAAG AGTCCACTAT TAAAGAACGT GGACTCCAAC 1801 GTCAAAGGGC GAAAAACCGT CTATCAGGGC GATGGCCCAC TACGTGAACC ATCACCCTAA 1861 TCAAGTTTTT TGGGGTCGAG GTGCCGTAAA GCACTAAATC GGAACCCTAA AGGGAGCCCC 1921 CGATTTAGAG CTTGACGGGG AAAGCCGGCG AACGTGGCGA GAAAGGAAGG GAAGAAAGCG 1981 AAAGGAGCGG GCGCTAGGGC GCTGGCAAGT GTAGCGGTCA CGCTGCGCGT AACCACCACA 2041 CCCGCCGCGC TTAATGCGCC GCTACAGGGC GCGTCAGGTG GCACTTTTCG GGGAAATGTG 2101 CGCGGAACCC CTATTTGTTT ATTTTTCTAA ATACATTCAA ATATGTATCC GCTCATGAGA 2161 CAATAACCCT GATAAATGCT TCAATAATAT TGAAAAAGGA AGAGTCCTGA GGCGGAAAGA 2221 ACCAGCTGTG GAATGTGTGT CAGTTAGGGT GTGGAAAGTC CCCAGGCTCC CCAGCAGGCA 2281 GAAGTATGCA AAGCATGCAT CTCAATTAGT CAGCAACCAG GTGTGGAAAG TCCCCAGGCT 2341 CCCCAGCAGG CAGAAGTATG CAAAGCATGC ATCTCAATTA GTCAGCAACC ATAGTCCCGC 2401 CCCTAACTCC GCCCATCCCG CCCCTAACTC CGCCCAGTTC CGCCCATTCT CCGCCCCATG 2461 GCTGACTAAT TTTTTTTATT TATGCAGAGG CCGAGGCCGC CTCGGCCTCT GAGCTATTCC 2521 AGAAGTAGTG AGGAGGCTTT TTTGGAGGCC TAGGCTTTTG CAAAGATCGA TCAAGAGACA 2581 GGATGAGGAT CGTTTCGCAT GATTGAACAA GATGGATTGC ACGCAGGTTC TCCGGCCGCT 2641 TGGGTGGAGA GGCTATTCGG CTATGACTGG GCACAACAGA CAATCGGCTG CTCTGATGCC 2701 GCCGTGTTCC GGCTGTCAGC GCAGGGGCGC CCGGTTCTTT TTGTCAAGAC CGACCTGTCC 2761 GGTGCCCTGA ATGAACTGCA AGACGAGGCA GCGCGGCTAT CGTGGCTGGC CACGACGGGC 2821 GTTCCTTGCG CAGCTGTGCT CGACGTTGTC ACTGAAGCGG GAAGGGACTG GCTGCTATTG 2881 GGCGAAGTGC CGGGGCAGGA TCTCCTGTCA TCTCACCTTG CTCCTGCCGA GAAAGTATCC 2941 ATCATGGCTG ATGCAATGCG GCGGCTGCAT ACGCTTGATC CGGCTACCTG CCCATTCGAC 3001 CACCAAGCGA AACATCGCAT CGAGCGAGCA CGTACTCGGA TGGAAGCCGG TCTTGTCGAT 3061 CAGGATGATC TGGACGAAGA GCATCAGGGG CTCGCGCCAG CCGAACTGTT CGCCAGGCTC 3121 AAGGCGAGCA TGCCCGACGG CGAGGATCTC GTCGTGACCC ATGGCGATGC CTGCTTGCCG 3181 AATATCATGG TGGAAAATGG CCGCTTTTCT GGATTCATCG ACTGTGGCCG GCTGGGTGTG 3241 GCGGACCGCT ATCAGGACAT AGCGTTGGCT ACCCGTGATA TTGCTGAAGA GCTTGGCGGC 3301 GAATGGGCTG ACCGCTTCCT CGTGCTTTAC GGTATCGCCG CTCCCGATTC GCAGCGCATC 3361 GCCTTCTATC GCCTTCTTGA CGAGTTCTTC TGAGCGGGAC TCTGGGGTTC GAAATGACCG 3421 ACCAAGCGAC GCCCAACCTG CCATCACGAG ATTTCGATTC CACCGCCGCC TTCTATGAAA 3481 GGTTGGGCTT CGGAATCGTT TTCCGGGACG CCGGCTGGAT GATCCTCCAG CGCGGGGATC 3541 TCATGCTGGA GTTCTTCGCC CACCCTAGGG GGAGGCTAAC TGAAACACGG AAGGAGACAA 3601 TACCGGAAGG AACCCGCGCT ATGACGGCAA TAAAAAGACA GAATAAAACG CACGGTGTTG 3661 GGTCGTTTGT TCATAAACGC GGGGTTCGGT CCCAGGGCTG GCACTCTGTC GATACCCCAC 3721 CGAGACCCCA TTGGGGCCAA TACGCCCGCG TTTCTTCCTT TTCCCCACCC CACCCCCCAA 3781 GTTCGGGTGA AGGCCCAGGG CTCGCAGCCA ACGTCGGGGC GGCAGGCCCT GCCATAGCCT 3841 CAGGTTACTC ATATATACTT TAGATTGATT TAAAACTTCA TTTTTAATTT AAAAGGATCT 3901 AGGTGAAGAT CCTTTTTGAT AATCTCATGA CCAAAATCCC TTAACGTGAG TTTTCGTTCC 3961 ACTGAGCGTC AGACCCCGTA GAAAAGATCA AAGGATCTTC TTGAGATCCT TTTTTTCTGC 4021 GCGTAATCTG CTGCTTGCAA ACAAAAAAAC CACCGCTACC AGCGGTGGTT TGTTTGCCGG 4081 ATCAAGAGCT ACCAACTCTT TTTCCGAAGG TAACTGGCTT CAGCAGAGCG CAGATACCAA 4141 ATACTGTCCT TCTAGTGTAG CCGTAGTTAG GCCACCACTT CAAGAACTCT GTAGCACCGC 4201 CTACATACCT CGCTCTGCTA ATCCTGTTAC CAGTGGCTGC TGCCAGTGGC GATAAGTCGT 4261 GTCTTACCGG GTTGGACTCA AGACGATAGT TACCGGATAA GGCGCAGCGG TCGGGCTGAA 4321 CGGGGGGTTC GTGCACACAG CCCAGCTTGG AGCGAACGAC CTACACCGAA CTGAGATACC 4381 TACAGCGTGA GCTATGAGAA AGCGCCACGC TTCCCGAAGG GAGAAAGGCG GACAGGTATC 4441 CGGTAAGCGG CAGGGTCGGA ACAGGAGAGC GCACGAGGGA GCTTCCAGGG GGAAACGCCT 4501 GGTATCTTTA TAGTCCTGTC GGGTTTCGCC ACCTCTGACT TGAGCGTCGA TTTTTGTGAT 4561 GCTCGTCAGG GGGGCGGAGC CTATGGAAAA ACGCCAGCAA CGCGGCCTTT TTACGGTTCC 4621 TGGCCTTTTG CTGGCCTTTT GCTCACATGT TCTTTCCTGC GTTATCCCCT GATTCTGTGG 4681 ATAACCGTAT TACCGCCATG CAT //
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pAmCyan1-N1质粒载体应用举例
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